Richard H. Ebright--Current Research Projects

Structural biology: x-ray crystallography

Structures of pathogen RNA polymerases
Escherichia coli RNA polymerase
Mycobacterium tuberculosis RNA polymerase
Staphylococcus aures RNA polymerase

Structures of transcription initiation complexes
RNA polymerase-promoter open complexes
RNA polymerase-promoter initial transcribing complexes

Single-molecule biophysics: fluorescence spectroscopy, nanomanipulation

Mechanism of transcription initiation
DNA loading
DNA unwinding
RNA polymerase clamp opening
RNA polymerase clamp closing
Sigma displacement
DNA scrunching in start-site selection
DNA scrunching in initial transcription

Mechanism of transcription elongation
RNA polymerase trigger-loop conformation
RNA polymerase clamp opening in pausing
DNA scrunching in pausing

Mechanism of transcription termination
RNA polymerase clamp opening in termination
RNA polymerase forward translocation in termination
RNA slippage in termination

Drug discovery: in vitro screening, in silico screening, medicinal chemistry

Identification and characterization of inhibitors of bacterial RNA polymerase
Myxopyronin
Lipiarmycin
Lariat peptides
Arylpropionyl-phloroglucinols
Aroyl-aryl-phenylalaninamides
Pseudouridimycin
Salinamide
GE23077
Engineering of biosynthetic loci for inhibitors of bacterial RNA polymerase
Myxopyronin biosynthetic locus
Lipiarmycin biosynthetic locus
Lariat-peptide biosynthetic loci
Synthesis of improved inhibitors of bacterial RNA polymerase
Myxopyronin analogs
Lipiarmycin analogs
Lariat-peptide analogs
Arylpropionyl-phloroglucinol analogs
Aroyl-aryl-phenylalaninamide analogs
Pseudouridimycin analogs
Bipartite inhibitors

Dr. Richard H. Ebright

Principal Investigator

Recent Publications

Robb, NC, Cordes T, Hwang L C, Gryte K, Duchi D, Craggs TD, Santoso Y, Weiss S, Ebright RH, Kapanidis AN. 2013. The transcription bubble of the RNA polymerase-promoter open complex exhibits conformational heterogeneity and millisecond-scale dynamics: implications for transcription start-site selection.. Journal of molecular biology. 425:875-885. Abstract

Bacterial transcription is initiated after RNA polymerase (RNAP) binds to promoter DNA, melts ~14 base-pairs around the transcription start site, and forms a single-stranded "transcription bubble" within a catalytically active RNAP-DNA open complex (RP(o)). There is significant flexibility in the transcription start site, which causes variable spacing between the promoter elements and the start site; this in turn causes differences in the length and sequence at the 5' end of RNA transcripts, and can be important for gene regulation. The start-site variability also implies the presence of some flexibility in the positioning of the DNA relative to the RNAP active site in RP(o). The flexibility may occur in the positioning of the transcription bubble prior to RNA synthesis and may reflect bubble expansion ("scrunching") or bubble contraction ("unscrunching"). Here, we assess the presence of dynamic flexibility in RP(o) with single-molecule Förster Resonance Energy Transfer. We obtain experimental evidence for dynamic flexibility in RP(o) using different FRET rulers and labelling positions. An analysis of FRET distributions of RP(o) using burst variance analysis reveals conformational fluctuations in RP(o) in the millisecond timescale. Further experiments using subsets of nucleotides and DNA mutations allowed us to reprogram the transcription start sites, in a way that can be described by repositioning of the single-stranded transcription bubble relative to the RNAP active site within RP(o). Our study marks the first experimental observation of conformational dynamics in the transcription bubble of RP(o) and indicates that DNA dynamics within the bubble affect the search for transcription start sites.

Chakraborty, A, Wang D, Ebright YW, Korlann Y, Kortkhonjia E, Kim T, Chowdhury S, Wigneshweraraj S, Irschik H, Jansen R et al.. 2012. Opening and closing of the bacterial RNA polymerase clamp.. Science (New York, N.Y.). 337(6094):591-5. AbstractWebsite

Using single-molecule fluorescence resonance energy transfer, we have defined bacterial RNA polymerase (RNAP) clamp conformation at each step in transcription initiation and elongation. We find that the clamp predominantly is open in free RNAP and early intermediates in transcription initiation but closes upon formation of a catalytically competent transcription initiation complex and remains closed during initial transcription and transcription elongation. We show that four RNAP inhibitors interfere with clamp opening. We propose that clamp opening allows DNA to be loaded into and unwound in the RNAP active-center cleft, that DNA loading and unwinding trigger clamp closure, and that clamp closure accounts for the high stability of initiation complexes and the high stability and processivity of elongation complexes.

Zhang, Y, Feng Y, Chatterjee S, Tuske S, Ho MX, Arnold E, Ebright RH. 2012. Structural Basis of Transcription Initiation.. Science (New York, N.Y.). 338(6110):1076-1080. AbstractWebsite

During transcription initiation, RNA polymerase (RNAP) binds and unwinds promoter DNA to form an RNAP-promoter open complex. We have determined crystal structures at 2.9 and 3.0 Å resolution of functional transcription initiation complexes comprising Thermus thermophilus RNA polymerase, σ(A), and a promoter DNA fragment corresponding to the transcription bubble and downstream dsDNA of the RNAP-promoter open complex. The structures show that σ recognizes the -10 element and discriminator element through interactions that include the unstacking and insertion into pockets of three DNA bases, and that RNAP recognizes the -4/+2 region through interactions that include the unstacking and insertion into a pocket of the +2 base. The structures further show that interactions between σ and template-strand ssDNA preorganize template-strand ssDNA to engage the RNAP active center.

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